DNA宏条形码技术在食草动物食性研究中的应用

随着测序技术的快速发展,整合DNA条形码和高通量测序的DNA宏条形码技术已经成为当前研究热点之一,在食草动物的食性鉴定中有很大潜力.放牧动物食性研究是动物营养学和草地生态学领域的重要研究内容.而与传统食性研究方法相比,宏条形码技术可通过对植物DNA条形码的高通量测序,获得样本中的物种组成进而分析动物食性.介绍了传统食性...     

RAPD分析与ITS序列分析在拟茎点霉分类鉴定上的应用

采用随机扩增多态性DNA(RAPD)技术和核糖体RNA基因(rDNA)转录间隔区(ITS)序列分析对22种拟茎点霉共34个菌株进行了系统发育研究。RAPD分析构建的UPGMA聚类图所反映的种间、种内关系与形态学分类结果基本一致,可以清楚地将分自7科寄主植物上的不同的种分别区分开来,但分自同科或同属寄主植物上的不同的种并不具有相近的亲缘关系。ITS序列分析结果不支持将Phomopsis mangiferae Ahmad和P. cytosporella Penz. et Sacc. 合并为同一个种的观点,同时还显示出P. mangiferae与P. psidii de Camara的亲缘关系非常近, 可能是异名同物;而为害木棉叶的拟茎点霉与杨梅枝枯病菌P. myricae Y.J.Huang et P.K.Chi之间的碱基差异亦属于种下不同菌株间的正常差别范围,很可能就是同一个种。对相同的供试菌株两技术所反映的亲缘关系趋势相同,表明两技术用于拟茎点霉的亲缘关系分析和种类鉴定均是可行的。     

RAPD分析与ITS序列分析在拟茎点霉分类鉴定上的应用*

采用随机扩增多态性DNA(RAPD)技术和核糖体RNA基因(rDNA)转录间隔区(ITS)序列分析对22种拟茎点霉共34个菌株进行了系统发育研究。RAPD分析构建的UPGMA聚类图所反映的种间、种内关系与形态学分类结果基本一致,可以清楚地将分自7科寄主植物上的不同的种分别区分开来,但分自同科或同属寄主植物上的不同的种并不具有相近的亲缘关系。ITS序列分析结果不支持将Phomopsis mangiferae Ahmad和P. cytosporella Penz. et Sacc. 合并为同一个种的观点,同时还显示出P. mangiferae与P. psidii de Camara的亲缘关系非常近, 可能是异名同物;而为害木棉叶的拟茎点霉与杨梅枝枯病菌P. myricae Y.J.Huang et P.K.Chi之间的碱基差异亦属于种下不同菌株间的正常差别范围,很可能就是同一个种。对相同的供试菌株两技术所反映的亲缘关系趋势相同,表明两技术用于拟茎点霉的亲缘关系分析和种类鉴定均是可行的。     

Visual clone identification of Penicillium commune isolates

A method for visual clone identification of Penicillium commune isolates was developed. The method is based on images of fungal colonies acquired after growth on a standard medium and involves a high degree of objectivity, which in future studies will make it possible for non-experts to perform a qualified identification of different species as well as clones within a species. A total of 77 P. commune isolates from a cheese dairy were 3-point inoculated on Yeast Extract Sucrose (YES) agar and incubated for 7 days at 25 degrees C. After incubation, the isolates were classified into groups containing the same genotype determined by DNA fingerprinting (AFLP). Each genotype also has a specific phenotype such as different colony colours. By careful image acquisition, colours were measured in a reproducible way. Prior to image analysis, each image was corrected with respect to colour, geometry and self-illumination, thereby gaining a set of directly comparable images. A method for automatic extraction of a given number of concentric regions was used. Using the positions of the regions, a number of relevant features--capturing colour and colour-texture from the surface of the fungal colonies--was extracted for further analysis. We introduced the Jeffreys-Matusitas (JM) distance between the feature distributions to express the similarity between regions in two colonies, and to evaluate the overall (weighted) similarity. The nearest neighbour (NN) classification rule was used. On a dataset from 137 isolates, we obtained a "leave-one-out" cross-validation identification rate of approximately 93-98% compared with the result of DNA fingerprinting.     

Peptostreptococcus vaginalis or Peptostreptococcus prevotii? Identification and clinical importance of a new species of Peptostreptococcus

Peptostreptococcus vaginalis is a recently described species of Gram-positive anaerobic coccus. We report one case in which P. vaginalis was isolated in pure culture from an abscess on the upper arm, and summarise nine further cases where it was isolated in mixed culture from other superficial sites, particularly infected leg ulcers. We suggest that clinical strains of P. vaginalis have probably been described in the past as Peptostreptococcus prevotii, a species which has frequently been reported from clinical surveys of anaerobic infections; their relative importance and appropriate treatment are discussed. Preformed enzyme profiles provide a simple method of identification accessible to routine diagnostic laboratories; when clinically significant isolates of GPAC are isolated in pure growth, they should be identified to the species level by use of preformed enzyme kits.     

Herbivory and tree mortality across a pinyon pine hybrid zone

We examined the abundances of three common insect herbivores on pure and hybrid pinyon pines along a 250-km transect in west-central Arizona, United States. Using six morphological traits, we developed a hybrid index to classify trees as pure Pinus californiarum, hybrid, or pure Pinus edulis. The insects (the stem-boring moth, Dioryctria albovittella, the scale insect, Matsucoccus acalyptus, and several species of pitch moths that produce wounds on the trunk and branches) exhibited different distributional patterns across tree types. Stem-boring moths were significantly more abundant on trees at hybrid sites compared to trees at pure sites. In addition, within hybrid sites, hybrids supported significantly more moth larvae than pure trees of either species. These two patterns support the hybrid susceptibility hypothesis in which hybrid breakdown results in increased susceptibility to herbivory. In contrast to stem-borers, there were significantly more pitch moth wounds on trees at pure P. californiarum sites than at hybrid and pure P. edulis sites. Within the hybrid zone, pitch moth abundance was equal on pure P. californiarum and hybrids, and both were significantly greater than on pure P. edulis. These within-site comparisons support the dominance hypothesis where hybrid resistance differs from one tree species, but not the other. Scale insects exhibited the most restricted distribution; over the 250 km transect they were found only in the hybrid zone. This supports the hybrid susceptibility and/or the stress hypothesis (i.e., species at the edge of their range suffer greater stress and are more susceptible to herbivory). We summed the mean numbers of these three common herbivores across sites and found that hybrid sites supported 2.1 and 3.9 times more herbivores than pure P. californiarum and P. edulis sites, respectively. Furthermore, tree mortality was on average, 35 times greater within the hybrid zone compared to pure zones of each species and was associated with the cumulative abundance of herbivores (r ²=0.646). Regardless of whether this mortality is due to insect infestation, stress or a combination of both, these results suggest that hybrid zones are important arenas of natural selection.     

Molecular differentiation and phylogeny of entomopathogenic nematodes (rhabditida: heterorhabditidae) based on ND4 gene sequences of Mitochondrial DNA.

We determined partial ND4 gene sequences of mitochondrial DNA from 15 heterorhabditid nematode isolates, representing 5 species collected from different regions of the world, by using polymerase chain reaction (PCR) and direct-sequencing of PCR products. Aligned nucleotide as well as amino acid sequences were used to differentiate nematode species by comparing sequence divergence and to infer phylogeny of the nematodes by using maximum parsimony and likelihood methods. Robustness of our phylogenetic trees was checked by bootstrap tests. The 15 nematode isolates can be divided into 7 haplotypes based on DNA sequences. On a larger scale, the sequence divergence revealed 4 distinct groups corresponding to 4 described species. No sequence divergence was detected from 5 isolates of Heterorhabditis bacteriophora or between Heterorhabditis marelatus to Heterorhabditis hepialius. Our sequence data yielded phylogenetic trees with identical topologies when different tree-building methods were used. Most relationships were also confirmed by using amino acid sequences in maximum parsimony analysis. Our molecular phylogeny of Heterorhabditis species support an existing taxonomy that is based largely on morphology and the sequence divergence of the ND4 gene permits species identification.     

PCR amplification of species-specific DNA sequences can distinguish among Phytophthora species.

We used PCR to differentiate species in the genus Phytophthora, which contains a group of devastating plant pathogenic fungi. We focused on Phytophthora parasitica, a species that can infect solanaceous plants such as tomato, and on Phytophthora citrophthora, which is primarily a citrus pathogen. Oligonucleotide primers were derived from sequences of a 1,300-bp P. parasitica-specific DNA segment and of an 800-bp P. citrophthora-specific segment. Under optimal conditions, the primers developed for P. parasitica specifically amplified a 1,000-bp sequence of DNA from isolates of P. parasitica. Primers for P. citrophthora similarly and specifically amplified a 650-bp sequence of DNA from isolates of P. citrophthora. Detectable amplification of these specific DNA sequences required picogram quantities of chromosomal DNA. Neither pair of primers amplified these sequences with DNAs from other species of Phytophthora or from the related genus Pythium. DNAs from P. parasitica and P. citrophthora growing in infected tomato stem tissue were amplified as distinctly as DNAs from axenic cultures of each fungal species. This is the first report on PCR-driven amplification with Phytophthora species-specific primers.     

Seeing is believing? Comparing plant–herbivore networks constructed by field co‐occurrence and DNA barcoding methods for gaining insights into network structures

Plant–herbivore interaction networks provide information about community organization. Two methods are currently used to document pairwise interactions among plants and insect herbivores. One is the traditional method that collects plant–herbivore interaction data by field observation of insect occurrence on host plants. The other is the increasing application of newly developed molecular techniques based on DNA barcodes to the analysis of gut contents. The second method is more appealing because it documents realized interactions. To construct complete interaction networks, each technique of network construction is urgent to be assessed. We addressed this question by comparing the effectiveness and reliability of the two methods in constructing plant–Lepidoptera larval network in a 50 ha subtropical forest in China. Our results showed that the accuracy of diet identification by observation method increased with the number of observed insect occurrences on food plants. In contrast, the molecular method using three plant DNA markers were able to identify food residues for 35.6% larvae and correctly resolved 77.3% plant (diet) species. Network analysis showed molecular networks had threefold more unique host plant species but fewer links than the traditional networks had. The molecular method detected plants that were not sampled by the traditional method, for example, bamboos, bryophytes and lianas in the diets of insect herbivores. The two networks also possessed significantly different structural properties. Our study indicates the traditional observation of co‐occurrence is inadequate, while molecular method can provide higher species resolution of ecological interactions.     

DNA barcoding of oomycetes with cytochrome c oxidase subunit I and internal transcribed spacer

Oomycete species occupy many different environments and many ecological niches. The genera Phytophthora and Pythium for example, contain many plant pathogens which cause enormous damage to a wide range of plant species. Proper identification to the species level is a critical first step in any investigation of oomycetes, whether it is research driven or compelled by the need for rapid and accurate diagnostics during a pathogen outbreak. The use of DNA for oomycete species identification is well established, but DNA barcoding with cytochrome c oxidase subunit I (COI) is a relatively new approach that has yet to be assessed over a significant sample of oomycete genera. In this study we have sequenced COI, from 1205 isolates representing 23 genera. A comparison to internal transcribed spacer (ITS) sequences from the same isolates showed that COI identification is a practical option; complementary because it uses the mitochondrial genome instead of nuclear DNA. In some cases COI was more discriminative than ITS at the species level. This is in contrast to the large ribosomal subunit, which showed poor species resolution when sequenced from a subset of the isolates used in this study. The results described in this paper indicate that COI sequencing and the dataset generated are a valuable addition to the currently available oomycete taxonomy resources, and that both COI, the default DNA barcode supported by GenBank, and ITS, the de facto barcode accepted by the oomycete and mycology community, are acceptable and complementary DNA barcodes to be used for identification of oomycetes.     

M13-microsatellite PCR and rDNA sequence markers for identification of Trichoderma (Hypocreaceae) species in Saudi Arabian soil

Seven fungal isolates were identified as pan-global Hypocrea/Trichoderma species, from section Trichoderma, on the basis of their morphology. These species were H. lixii/T. harzianum and H. orientalis/T. longibrachiatum. PCR-based markers with primer M13 (core sequence of phage M13) and internal-transcribed spacer sequences of ribosomal DNA were used to confirm the identity of the two Trichoderma species. Sequence identification was performed using the TrichOKEY version 2.0 barcode program and the multilocus similarity search database TrichoBLAST. Sequences from the ribosomal DNA internal-transcribed spacer regions showed limited variation among the Trichoderma species. This analysis divided the isolates into two main groups. Grouping the isolates based on cluster analysis of their DNA profiles matched the grouping based on morphological taxonomy. Molecular data obtained from analyses of gene sequences are essential to distinguish phonetically cryptic species in this group and to establish phylogenetic relationships.     

Genotypic analyses of lactobacilli with a range of tannase activities isolated from human feces and fermented foods

A total of 77 tannase producing lactobacilli strains isolated from human feces or fermented foods were examined for their genotypic profiles and intensities of tannase production. With a PCR-based assay targeting recA gene, all strains except one isolate were assigned to either Lactobacillus plantarum, L. paraplantarum, or L. pentosus whereas a 16/23S rDNA targeted PCR-based assay identified all except 6 isolates (inclusive of the above one isolate) as one of the closely related species. Subsequent DNA/DNA hybridization assays revealed that these 6 exceptional isolates showed low homology (between 1.2% and 55.8% relative DNA binding) against type strains of the three species. Supplemental carbohydrate fermentation profiles on the 6 isolates indicated that two of them were identified as L. acidophilus, one as Pediococcus acidilactici, one as P. pentosaceus, and two remained unidentifiable. The evidence suggests that the 16/23S rDNA targeted PCR assay can be used as a reliable identification tool for the closely related lactobacilli, and that the tannase gene is widely distributed within members of the Lactobacillaceae family. Meanwhile, a randomly amplified polymorphism DNA (RAPD) analysis revealed that all except 8 isolates were well allocated in 4 major RAPD clusters, though not species specific, consisting of two L. plantarum predominant clusters, one L. paraplantarum predominant, and one L. pentosus predominant. The RAPD patterns of the 8 non-clustered isolates, which consisted of the 6 unidentifiable isolates and 2 isolates identified as L. pentosus, were <40% similarity to those belonging to the 4 clusters. A quantitative assay of the tannase activities showed that there was a marked variation in the activities among the strains, which did not correlate with either species identification or clustering by RAPD.     

Oligonucleotide array for identification and detection of pythium species

A DNA array containing 172 oligonucleotides complementary to specific diagnostic regions of internal transcribed spacers (ITS) of more than 100 species was developed for identification and detection of Pythium species. All of the species studied, with the exception of Pythium ostracodes, exhibited a positive hybridization reaction with at least one corresponding species-specific oligonucleotide. Hybridization patterns were distinct for each species. The array hybridization patterns included cluster-specific oligonucleotides that facilitated the recognition of species, including new ones, belonging to groups such as those producing filamentous or globose sporangia. BLAST analyses against 500 publicly available Pythium sequences in GenBank confirmed that species-specific oligonucleotides were unique to all of the available strains of each species, of which there were numerous economically important ones. GenBank entries of newly described species that are not putative synonyms showed no homology to sequences of the spotted species-specific oligonucleotides, but most new species did match some of the cluster-specific oligonucleotides. Further verification of the specificity of the DNA array was done with 50 additional Pythium isolates obtained by soil dilution plating. The hybridization patterns obtained were consistent with the identification of these isolates based on morphology and ITS sequence analyses. In another blind test, total DNA of the same soil samples was amplified and hybridized on the array, and the results were compared to those of 130 Pythium isolates obtained by soil dilution plating and root baiting. The 13 species detected by the DNA array corresponded to the isolates obtained by a combination of soil dilution plating and baiting, except for one new species that was not represented on the array. We conclude that the reported DNA array is a reliable tool for identification and detection of the majority of Pythium species in environmental samples. Simultaneous detection and identification of multiple species of soilborne pathogens such as Pythium species could be a major step forward for epidemiological and ecological studies.     

Rapid identification of Phytophthora ramorum using PCR-SSCP analysis of ribosomal DNA ITS-1

AIMS: The primary objectives of this study were to determine if a single-strand conformation polymorphism (SSCP) analysis can be used for rapid identification of Phytophthora ramorum, an important quarantine plant pathogen worldwide, and to further assess the potential of the SSCP technique as a taxonomic tool for the genus Phytophthora. METHODS AND RESULTS: SSCP of ribosomal DNA internal transcribed spacer 1 was characterized for 12 isolates of P. ramorum, using a recently reported protocol. The SSCP patterns of this species then were compared with those of 18 closely related Phytophthora species. Phytophthora ramorum had a unique pattern and was easily distinguished from genetically, morphologically and ecologically close relatives. CONCLUSION: An immediate benefit of this study is provision of a highly effective and efficient identification tool for P. ramorum in the quarantine process. SIGNIFICANCE AND IMPACT OF THE STUDY: This study also provides additional evidence demonstrating that the SSCP is an ideal DNA marker for species differentiation within the genus Phytophthora.     

Pasteurella pneumotropica and the prevalence of the AHP (Actinobacillus, Haemophilus, Pasteurella)-group in laboratory animals

134 bacterial isolates originally identified as Pasteurella pneumotropica were cultured from healthy, ill or dead mice, rats, hamsters, guinea pigs, rabbits and cats originating from 7 conventional laboratory animal facilities. They occurred seldom in pure culture and were found in a variety of organs. Thorough identification (41 criteria) revealed that only 83 isolates (62%) were P. pneumotropica and could be subdivided into 3 biotypes. 3 isolates were P. aerogenes, 1 was P. ureae, 11 (8%) were qualified as Actinobacillus spp. and 13 (10%) as Haemophilus spp. Close relationship of the 3 genera--the 'AHP-group' --made the differentiation difficult. 23 atypical cultures were discarded at the beginning of the study as not belonging to the 'AHP-group'. Two-thirds of isolates were associated with inflammation or suppuration; Haemophilus spp. seemed to be more pathogenic than Pasteurella and Actinobacillus species.     

Direct identification of bacteria in clinical respiratory samples using fluorescent amplicon length analysis of 16S-23S rRNA spacer-region

We describe the development and application of a rapid and universal molecular technique for direct identification of multiple bacteria in clinical samples. Amplification of the 16S-23S rRNA spacer-region using universal primers led to fragment patterns distinct for different bacterial species and that were analyzed with fluorescent amplicon length analysis (FALA). 136 pure cultures of clinical isolates and 20 culture collection strains belonging to 22 different medically important species were used to create a primary database of fragments with sizes between 100 and 1000 bp. Subsequently, 127 respiratory samples were analyzed with culture-based techniques and via FALA of the 16S-23S rRNA spacer-region. Two DNA extraction methods were evaluated: Instagene (FALA-I) and Fastprept (FALA-P). Of the 127 samples, 26 culture-negative samples were also negative with FALA-P. Of 18 samples with growth of commensal oral flora, 10 gave a mixed oral flora pattern with FALA-P and 8 gave a negative result. For 54 samples with growth of a single bacterial species, FALA-P gave an identical result for 46. For 29 samples with growth of more than one bacterial species, identical results were obtained in 19 samples. False-negative results with FALA-P were mostly due to paucity (less than 10(3) CFU/ml) of bacteria (12 out of 18 false-negatives) or difficulties with homogenization of viscous samples (6 out of 18 false-negatives).With regard to identification of all significant pathogens of clinical samples tested, the sensitivity of FALA-P was 77% and its specificity was 100%. With FALA-I, the number of false-negative results was higher than with FALA-P due to less efficient extraction of DNA, particularly with Staphylococcal species. FALA-P allows rapid and direct identification of multiple species directly from clinical samples; pauci-cellular samples may give false-negative results.     

Genetic comparison of Bacillus anthracis and its close relatives using amplified fragment length polymorphism and polymerase chain reaction analysis

Amplified fragment length polymorphism (AFLP) analysis allows a rapid, relatively simple analysis of a large portion of a microbial genome, providing information about the species and its phylogenetic relationship to other microbes (Vos et al. 1995). The method simply surveys the genome for length and sequence polymorphisms. The AFLP pattern identified can be used for comparison to the genomes of other species. Unlike other methods, it does not rely on analysis of a single genetic locus that may bias the interpretation of results and does not require any prior knowledge of the targeted organism. Moreover, a standard set of reagents can be applied to any species without using species-specific information or molecular probes. We are using AFLP analysis to rapidly identify different bacterial species. A comparison of AFLP profiles generated from a large battery of Bacillus anthracis strains shows very little variability among different isolates (Keim et al. 1997). By contrast, there is a significant difference between AFLP profiles generated for any B. anthracis strain and even the most closely related Bacillus species. Sufficient variability is apparent among all known microbial species to allow phylogenetic analysis based on large numbers of genetically unlinked loci. These striking differences among AFLP profiles allow unambiguous identification of previously identified species and phylogenetic placement of newly characterized isolates relative to known species based on a large number of independent genetic loci. Data generated thus far show that the method provides phylogenetic analyses that are consistent with other widely accepted phylogenetic methods. However, AFLP analysis provides a more detailed analysis of the targets and samples a much larger portion of the genome. Consequently, it provides an inexpensive, rapid means of characterizing microbial isolates to further differentiate among strains and closely related microbial species. Such information cannot be rapidly generated by other means. AFLP sample analysis quickly generates a very large amount of molecular information about microbial genomes. However, this information cannot be analysed rapidly using manual methods. We are developing a large archive of electronic AFLP signatures that is being used to identify isolates collected from medical, veterinary, forensic and environmental samples. We are also developing the computational packages necessary to rapidly and unambiguously analyse the AFLP profiles and conduct a phylogenetic comparison of these data relative to information already in our database. We will use this archive and the associated algorithms to determine the species identity of previously uncharacterized isolates and place them phylogenetically relative to other microbes based on their AFLP signatures. This study provides significant new information about microbes with environmental, veterinary and medical significance. This information can be used in further studies to understand the relationships among these species and the factors that distinguish them from one another. It should also allow the identification of unique factors that contribute to important microbial traits, including pathogenicity and virulence. We are also using AFLP data to identify, isolate and sequence DNA fragments that are unique to particular microbial species and strains. The fragment patterns and sequence information provide insights into the complexity and organization of bacterial genomes relative to one another. They also provide the information necessary for the development of species-specific polymerase chain reaction primers that can be used to interrogate complex samples for the presence of B. anthracis, other microbial pathogens or their remnants.     

Molecular typing of Pasteurella pneumotropica isolated from rodents by amplified 16S ribosomal DNA restriction analysis and pulsed-field gel electrophoresis

A total of 52 isolates of Pasteurella pneumotropica obtained from rodents were examined for their genetic heterogeneity. On the basis of DNA restriction analysis, including amplified 16S ribosomal DNA restriction analysis (ARDRA) and pulsed-field gel electrophoresis (PFGE), differences were identified among the isolates. ARDRA typing with Hae III revealed 4 different banding patterns of the P. pneumotropica isolates. Eighty-two percent of the 23 isolates identified as a-1 were derived from mice, whereas all the isolates identified as a-3 were derived from rats. Most of the isolates, which showed hemolytic activity on blood agar, obtained from mice and rats, were identified as a-2 and a-4, respectively. By restriction analysis of genomic DNA, Apa I and Not I digestion differentiated 9 variants and an undiscriminating group. However, no close relation with regard to the phenotypic characteristics was observed among the variants. The isolates identified as a-2 and a-4 could not be distinguished by PFGE analysis. DNA restriction analysis revealed that the genetic diversity of the P. pneumotropica isolates was more complex than the phenotypic characteristics among the species, and that at least the P. pneumotropica isolates were clearly differentiated into 4 groups by ARDRA typing with Hae III.     

Rapid identification of marine bioluminescent bacteria by amplified 16S ribosomal RNA gene restriction analysis

To rapidly identify natural isolates of marine bioluminescent bacteria, we developed amplified ribosomal DNA restriction analysis (ARDRA) methods. ARDRA, which is based on the restriction patterns of 16S rRNA gene digested with five enzymes (EcoRI, DdeI, HhaI, HinfI, RsaI), clearly distinguished the 14 species of marine bioluminescent bacteria currently known, which belong to the genera Vibrio, Photobacterium, and Shewanella. When we applied ARDRA to 129 natural isolates from two cruises in Sagami Bay, Japan, 127 were grouped into six ARDRA types with distinctive restriction patterns; these isolates represented the bioluminescent species, P. angustum, P. leiognathi, P. phosphoreum, S. woodyi, V. fischeri, and V. harveyi. The other two isolates showing unexpected ARDRA patterns turned out to have 16S rRNA gene sequences similar to P. leiognathi and P. phosphoreum. Nevertheless, ARDRA provides a simple and fairly robust means for rapid identification of the natural isolates of marine bioluminescent bacteria, and is therefore useful in studying their diversity.